Quantification of 4-Heptanone in Urine by Headspace GC-MS Analysis Using a Multipurpose Sampler

Importance of the topic

The non-invasive analysis of volatile organic compounds in urine provides valuable insights into human metabolic status. 4-Heptanone is a diagnostic ketone linked to metabolic disorders, including diabetes mellitus. Reliable quantification of this biomarker can support clinical and research applications in metabolic profiling.

Study overview and objectives

This study evaluates a fully automated headspace GC-MS method using a Gerstel Multi Purpose Sampler (MPS) for profiling urinary volatiles and quantifying 4-heptanone. A pilot comparison of diabetic patients (Type I and II) and healthy controls aimed to assess method performance and explore potential clinical correlations.

Methodology and instrumentation

Sample preparation and headspace procedure:

• Collected 139 urine samples (51 diabetic, 42 controls); acidified (30 µL conc. HCl) and 1 mL aliquots in 2 mL vials.
• Preheating: five-position module at 70 °C for 10 min; headspace syringe at 70 °C.
• Headspace sampling: helium-flushed 1 000 µL gas-tight syringe, controlled volume and injection depth.
• Cold trapping and focusing: CIS-3 cooled to –50 °C (condition II) or 10 °C (I), then heated to 300 °C at 12 °C/s and transferred to GC.

GC-MS conditions:

• Columns: 60 m DB-5, di 0.25 mm, df 0.25 µm; or 30 m HP-5 MS, di 0.25 mm, df 0.33 µm.
• Carrier gas: helium at 30–100 kPa; split 30:1 or splitless (0.1–1.1 min).
• Oven programs: 60→100 °C @5 °C/min→240 °C @25 °C/min (I); 35 °C (2 min)→260 °C @15 °C/min (II).
• MSD: scan 10–260 amu; interface at 280 °C.

Key results and discussion

Volatile profiling:

• Identified 20+ compounds including methanethiol, dimethylsulfide, 2-pentanone, toluene, allyl isothiocyanate, and 4-heptanone.
• Acidification increased peak count, revealing additional sulfur and heterocyclic compounds.

4-Heptanone quantification:

• Calibration linear over 40–800 ng/mL (r=0.999); intra-assay CV 3.0–3.4%.
• Pilot study medians: diabetic group 179 ng/mL (17–978 ng/mL), controls 188 ng/mL (27–1044 ng/mL).
• No significant difference between groups; a few controls exhibited very high levels (up to 3720 ng/mL), suggesting potential environmental sources (e.g., plasticizer metabolism).

Benefits and practical applications

The automated headspace GC-MS workflow offers:

• High throughput for routine clinical or quality-control laboratories.
• Enhanced sensitivity and precision for volatile ketone biomarkers.
• Cost savings via a versatile sampler capable of liquid, gas, and headspace modes.

Future trends and potential applications

• Investigate environmental vs. endogenous origins of urinary 4-heptanone.
• Expand volatile biomarker panels for metabolic and disease profiling.
• Integrate with pattern recognition and multivariate statistics to improve diagnostic specificity.
• Advance cold-trap and sampling technologies for lower detection limits.

Conclusion

The described headspace GC-MS method utilizing a Gerstel MPS and CIS-3 offers robust, sensitive, and automated analysis of urinary volatiles. It is well suited for metabolic profiling and routine clinical applications, with demonstrated precision for 4-heptanone quantification.

Instrumentation

• Gerstel Multi Purpose Sampler (headspace mode) with 1 000 µL gas-tight syringe and pre-heating module.
• Gerstel CIS-3 cold injection system (–50 °C to 300 °C).
• HP-5890 GC and HP-5972 MSD.
• Columns: Supelco Carbotrap 20 mm liner; DB-5 (60 m) and HP-5 MS (30 m).

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